蛋白质纯化手册

ProteinPurificationHandbook18-1132-29EditionABHiTrap,Sepharose,STREAMLINE,Sephadex,MonoBeads,MonoQ,MonoS,MiniBeads,RESOURCE,SOURCE,Superdex,Superose,HisTrap,HiLoad,HiPrep,INdEX,BPG,BioProcess,FineLINE,MabTrap,MAbAssistant,Multiphor,FPLC,PhastSystemandÄKTAaretrademarksofAmershamPharmaciaBiotechLimitedoritssubsidiaries.AmershamisatrademarkofNycomedAmershamplcPharmaciaandDropDesignaretrademarksofPharmacia&UpjohnIncCoomassieisatrademarkofICIplcAllgoodsandservicesaresoldsubjecttothetermsandconditionsofsaleofthecompanywithintheAmershamPharmaciaBiotechgroupwhichsuppliesthem.Acopyofthesetermsandconditionsofsaleisavailableonrequest.©AmershamPharmaciaBiotechAB1999-Allrightsreserved.AmershamPharmaciaBiotechABSE-75184UppsalaSwedenAmershamPharmaciaBiotechUKLimitedAmershamPlaceLittleChalfontBuckinghamshireEnglandHP79NAAmershamPharmaciaBiotechInc800CentennialAvenuePOBox1327PiscatawayNJ08855USAProteinPurificationHandbookContentsIntroduction........................................................................................................7Chapter1PurificationStrategies-ASimpleApproach......................................................9Preparation............................................................................................10ThreePhasePurificationStrategy..........................................................10GeneralGuidelinesforProteinPurification............................................12Chapter2Preparation......................................................................................................13BeforeYouStart....................................................................................13SampleExtractionandClarification......................................................16Chapter3ThreePhasePurificationStrategy....................................................................19Principles................................................................................................19SelectionandCombinationofPurificationTechniques..........................20SampleConditioning..............................................................................26Chapter4Capture..................................................................................................29Chapter5IntermediatePurification........................................................................37Chapter6Polishing................................................................................................40Chapter7ExamplesofProteinPurificationStrategies............................................45Threesteppurificationofarecombinantenzyme..................................45Threesteppurificationofarecombinantantigenbindingfragment......49Twosteppurificationofamonoclonalantibody....................................54Onesteppurificationofanintegralmembraneprotein..........................57Chapter8StorageConditions................................................................................61ExtractionandClarificationProcedures................................................62Chapter9PrinciplesandStandardConditionsforPurificationTechniques............73Ionexchange(IEX)................................................................................73Hydrophobicinteraction(HIC)..............................................................79Affinity(AC)..........................................................................................85Gelfiltration(GF)..................................................................................88Reversedphase(RPC)............................................................................92Expandedbedadsorption(EBA)............................................................957IntroductionThedevelopmentoftechniquesandmethodsforproteinpurificationhasbeenanessentialpre-requisiteformanyoftheadvancementsmadeinbiotechnology.Thisbookletprovidesadviceandexamplesforasmoothpathtoproteinpurification.Proteinpurificationvariesfromsimpleone-stepprecipitationprocedurestolargescalevalidatedproductionprocesses.Oftenmorethanonepurificationstepisnecessarytoreachthedesiredpurity.Thekeytosuccessfulandefficientproteinpurificationistoselectthemostappropriatetechniques,optimisetheirperformancetosuittherequirementsandcombinetheminalogicalwaytomaximiseyieldandminimisethenumberofstepsrequired.Mostpurificationschemesinvolvesomeformofchromatography.Asaresultchromatographyhasbecomeanessentialtoolineverylaboratorywhereproteinpurificationisneeded.Theavailabilityofdifferentchromatographytechniqueswithdifferentselectivitiesprovidesapowerfulcombinationforthepurificationofanybiomolecule.RecombinantDNAdevelopmentsoverthepastdecadehaverevolutionisedtheproductionofproteinsinlargequantities.Proteinscanevenbeproducedinformswhichfacilitatetheirsubsequentchromatographicpurification.However,thishasnotremovedallchallenges.Hostcontaminantsarestillpresentandproblemsrelatedtosolubility,structuralintegrityandbiologicalactivitycanstillexist.Althoughtheremayappeartobeagreatnumberofparameterstoconsider,wi